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- EMDB-4158: PilMN complex from Thermus thermophilus -

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Basic information

Entry
Database: EMDB / ID: EMD-4158
TitlePilMN complex from Thermus thermophilus
Map dataThermus PilMN complex
Sample
  • Complex: PilMN complex
    • Protein or peptide: PilN
    • Protein or peptide: PilM
Biological speciesThermus thermophilus HB8 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 27.0 Å
AuthorsDerrick JP / Collins RF
CitationJournal: Proc Natl Acad Sci U S A / Year: 2013
Title: Structure and assembly of an inner membrane platform for initiation of type IV pilus biogenesis.
Authors: Vijaykumar Karuppiah / Richard F Collins / Angela Thistlethwaite / Ya Gao / Jeremy P Derrick /
Abstract: Type IV pili are long fibers that are assembled by polymerization of a major pilin protein in the periplasm of a wide range of bacteria and archaea. They play crucial roles in pathogenesis, DNA ...Type IV pili are long fibers that are assembled by polymerization of a major pilin protein in the periplasm of a wide range of bacteria and archaea. They play crucial roles in pathogenesis, DNA transformation, and motility, and are capable of rapid retraction, generating powerful motor forces. PilN and PilO are integral inner membrane proteins that are essential for type IV pilus formation. Here, we show that PilN and PilO from Thermus thermophilus can be isolated as a complex with PilM, a cytoplasmic protein with structural similarities to the cytoskeletal protein MreB. The crystal structure of the periplasmic portion of PilN forms a homodimer with an extensive, conserved interaction interface. We conducted serial 3D reconstructions by electron microscopy of PilMN, PilMNO, and PilMNO bound to the major pilin protein PilA4, to chart the assembly of the inner membrane pilus biogenesis platform. PilN drives the dimerization of the PilMN complex with a stoichiometry of 2:2; binding of two PilO monomers then causes the PilN periplasmic domains to dissociate. Finally, two PilA4 monomers bind to the periplasmic domains of PilN and PilO, to generate a T-shaped complex that is primed for addition of the pilin to the nascent pilus fiber. Docking of structures for PilM, PilN, PilO, and PilA4 into the electron density maps of the transmembrane complexes was used to generate a sequence of molecular structures that chart the initial events in type IV pilus formation, and provide structural information on the early events in this important secretion process.
History
DepositionOct 20, 2016-
Header (metadata) releaseOct 26, 2016-
Map releaseOct 26, 2016-
UpdateOct 26, 2016-
Current statusOct 26, 2016Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.63
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.63
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4158.png

Downloads & links

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Map

FileDownload / File: emd_4158.map.gz / Format: CCP4 / Size: 1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThermus PilMN complex
Voxel sizeX=Y=Z: 3.49 Å
Density
Contour LevelBy AUTHOR: 0.63 / Movie #1: 0.63
Minimum - Maximum-0.20221405 - 1.9918385
Average (Standard dev.)0.029468894 (±0.17929818)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-2-2-2
Dimensions646464
Spacing646464
CellA=B=C: 223.36 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.493.493.49
M x/y/z646464
origin x/y/z0.0000.0000.000
length x/y/z223.360223.360223.360
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-2-2-2
NC/NR/NS646464
D min/max/mean-0.2021.9920.029

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Supplemental data

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Sample components

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Entire : PilMN complex

EntireName: PilMN complex
Components
  • Complex: PilMN complex
    • Protein or peptide: PilN
    • Protein or peptide: PilM

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Supramolecule #1: PilMN complex

SupramoleculeName: PilMN complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Thermus thermophilus HB8 (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pET28a

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Macromolecule #1: PilN

MacromoleculeName: PilN / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Thermus thermophilus HB8 (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MIRLNLLPKN LRRRVEPGWW RLLALLFALV VLGVLGLVHY TAYTELSLAK AERDQLQAEV EALRPFIAE LGRLQEERKA LEALLAIREG LEKNAVPWSQ YLAAFINQIP RAGGRLEVAL R SVSARALS EEEAARLAQE GTYDGKRIRV EFALQGEALS REALVRFIRA ...String:
MIRLNLLPKN LRRRVEPGWW RLLALLFALV VLGVLGLVHY TAYTELSLAK AERDQLQAEV EALRPFIAE LGRLQEERKA LEALLAIREG LEKNAVPWSQ YLAAFINQIP RAGGRLEVAL R SVSARALS EEEAARLAQE GTYDGKRIRV EFALQGEALS REALVRFIRA FETSPRFGIE FQ GASLDEG RGLYTFSARV GVTGGESGAR

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Macromolecule #2: PilM

MacromoleculeName: PilM / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Thermus thermophilus HB8 (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MFKSLSQLFR PRVEALGLEI GASALKLVEV SGNPPALKAL ASRPTPPGLL MEGMVAEPAA LAQEIKELL LEARTRKRYV VTALSNLAVI LRPIQVPKMP LKEMEEAVRW EAERYIPFPI D EVVLDFAP LTPLSEVQEG EQVQVMVAAA RQEAVAGVLE ALRGAGLVPV ...String:
MFKSLSQLFR PRVEALGLEI GASALKLVEV SGNPPALKAL ASRPTPPGLL MEGMVAEPAA LAQEIKELL LEARTRKRYV VTALSNLAVI LRPIQVPKMP LKEMEEAVRW EAERYIPFPI D EVVLDFAP LTPLSEVQEG EQVQVMVAAA RQEAVAGVLE ALRGAGLVPV VLDVKPFAGL YP LEARLAE EPDRVFLALD IGAESTSLVL LRGDKPLAVR VLTLSGKDFT EAIARSFNLD LLA AEEVKR TYGMATLPTE DEELLLDFDA ERERYSPGRI YDAIRPVLVE LTQELRRSLE FFRI QLEEA SPEVGYLLGG GSKLRGLASL LTDTLGVNFE PVNPWEAVAV DPKRFESEQL QEIGP EFAV ALGLALRGVE PLD GASLDE GRGLYTFSAR VGVTGGESGA R

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI POLARA 300
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Average exposure time: 1.0 sec. / Average electron dose: 25.0 e/Å2
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - details: EMAN
Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: PROJECTION MATCHING
Final reconstructionResolution.type: BY AUTHOR / Resolution: 27.0 Å / Resolution method: FSC 0.5 CUT-OFF / Number images used: 12074

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